CN113831296B

CN113831296B - 1-phenyl-3- (4- (pyrimidine-5-ylamino) phenyl) prop-2-en-1-one compounds and uses thereof

Info

Publication number: CN113831296B
Application number: CN202110849763.3A
Authority: CN
Inventors: 李博; 刘媛媛; 韩楚; 戴荣继; 邓玉林
Original assignee: Beijing Institute of Technology BIT
Current assignee: Beijing Institute of Technology BIT
Priority date: 2021-07-27
Filing date: 2021-07-27
Publication date: 2023-07-21
Anticipated expiration: 2041-07-27
Also published as: CN113831296A

Abstract

The invention provides a compound of a 1-phenyl-3- (4- (pyrimidine-5-ylamino) phenyl) prop-2-en-1-one compound or a pharmaceutically acceptable salt thereof with a chemical formula (I)Wherein R1, R2, R3 and R4 are each hydrogen or hydroxy, and R5 and R6 are C1-16 alkyl or C2-16 alkenyl. The compound has good inhibition effect on the aggregation of Abeta protein, protects the activity of nerve cells by inhibiting the aggregation of Abeta protein, and inhibits the death of cell iron by inhibiting the peroxidation level of cell lipid. It is expected to be further developed for treating Alzheimer's disease and the like, or treating diseases related to cell iron death as an iron death inhibitor.

Description

1-phenyl-3- (4- (pyrimidine-5-ylamino) phenyl) prop-2-en-1-one compounds and uses thereof

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a 1-phenyl-3- (4- (pyrimidine-5-ylamino) phenyl) prop-2-en-1-one compound, a preparation method and application thereof.

Background

Alzheimer's disease is a chronic, progressive and irreversible neurodegenerative disease first proposed by Alois Alzheimer in 1907. Despite the enormous efforts of drug development for AD over 100 years, there is no cure for this fatal disease. AD is characterized by irreversible memory loss, language impairment, severe behavioral abnormalities and learning impairment. To date, three types of acetylcholinesterase (AChE) inhibitors, donepezil, rivastigmine and galantamine and an NMDA receptor (NMDAR) antagonist memantine are approved by the united states Food and Drug Administration (FDA) for the treatment of AD. In addition, 11 months 2019, china national drug administration approved a new drug, sodium fructooligosaccharide (GV-971), developed by Shanghai Green cereal pharmaceutical Co., ltd. For improving cognitive dysfunction in patients with mild to moderate AD. The pathogenesis of sporadic AD is still unclear and has not yet been elucidated. Amyloid plaques formed by aggregation of Abeta proteins are the most important pathological features of AD in early stage, and aggregation of beta-amyloid in brain can generate neurotoxicity to cause death of nerve cells and promote worsening of AD diseases, and the beta-amyloid hypothesis is the main therapeutic target in the research and development of AD drugs at present, so that Abeta protein aggregation inhibition is determined as a core target of design drugs.

Recent studies have shown that AD is closely related to iron death. Iron death is a regulated cell death that was newly discovered in 12 years due to oxidative perturbation of the intracellular microenvironment controlled by glutathione peroxidase 4 (GPX 4) and can be inhibited by iron chelates and lipophilic antioxidants. Iron death is characterized by iron imbalance and lipid peroxide accumulation and an increase in Reactive Oxygen Species (ROS), which are also pathological features of AD. A number of AD clinical study drugs such as the free radical trapping antioxidant tocopherol and the iron chelator DFO proved to be iron death inhibitors. Thus, inhibition of iron death has become a new target for the treatment of AD. Neuroprotection strategies against intracellular ROS-mediated oxidative damage would be a meaningful way to treat AD. Therefore, we selected inhibition of cellular iron death and aβ protein aggregation caused by oxidative damage as targets for drug design and screening.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention provides a 1-phenyl-3- (4- (pyrimidine-5-ylamino) phenyl) prop-2-en-1-one compound, a preparation method and application thereof, has good inhibition effect on aggregation of Abeta protein and cell iron death, and is used for treating Alzheimer's disease and diseases related to cell iron death.

The invention provides the following technical scheme:

1-phenyl-3- (4- (pyrimidin-5-ylamino) phenyl) prop-2-en-1-one compounds or pharmaceutically acceptable salts thereof, characterized in that the compounds of formula (I),

wherein R1, R2, R3 and R4 are each hydrogenOr hydroxy, R5 and R6 are each C _1-16 Alkyl or C _2-16 Alkenyl groups.

Further, when R1 is hydrogen, R2 is hydroxy, R3 is hydrogen, R4 is hydrogen, R5 is methyl, R6 is methyl, the compound is (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (3-hydroxyphenyl) prop-2-en-1-one.

Further, when R1 is hydrogen, R2 is hydroxy, R3 is hydrogen, R4 is hydroxy, R5 is methyl, R6 is methyl, the compound is (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (3, 5-dihydroxyphenyl) prop-2-en-1-one.

Further, when R1 is hydroxy, R2 is hydroxy, R3 is hydroxy, R4 is hydrogen, R5 is methyl, R6 is methyl, the compound is (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (2, 3, 4-trihydroxyphenyl) prop-2-en-1-one.

Further, the application in preparing the medicine for preventing and treating the Alzheimer disease.

Further, use in the preparation of an inhibitor for cellular iron death.

A pharmaceutical composition comprising a compound and a pharmaceutically acceptable carrier or excipient.

A medicament having a therapeutic agent for alzheimer's disease, 1-phenyl-3- (4- (pyrimidin-5-ylamino) phenyl) prop-2-en-1-one compound, or solvates, stereoisomers, tautomers and prodrugs thereof.

A method of preparing a compound comprising the steps of:

step 1, carrying out aldehyde-ketone condensation reaction on acetophenone and p-bromobenzaldehyde to obtain chalcone compounds;

and (3) carrying out substitution reaction on the product obtained in the step (2) and 2, 5-diaminopyrimidine to obtain a 1-phenyl-3- (4- (pyrimidine-5-ylamino) phenyl) prop-2-en-1-one compound.

Further, if the acetophenone in the second step is 3, 5-dihydroxyacetophenone or 2,3, 4-trihydroxyacetophenone, the method further comprises step 0 before the step 1: reacting polyhydroxy acetophenone with bromomethyl methyl ether, and treating to obtain methoxymethyl-protected acetophenone;

and step 2, dropwise adding an HCI water solution into the product to react and remove the methoxymethyl group to obtain a target final product.

Further, in the step 0,3, 5-dihydroxyacetophenone or 2,3, 4-dihydroxyacetophenone is dissolved in anhydrous acetone, 3 times of potassium carbonate is added, 3 times of bromomethyl methyl ether is slowly added dropwise under the ice bath condition, then reflux reaction is carried out for 4 hours, and after cooling to room temperature, the methoxymethyl-protected acetophenone is obtained by treatment;

in the step 1, KOH aqueous solution with the concentration ratio of 10% is used as a catalyst, and after the reaction is carried out for 48 to 96 hours at normal temperature, HCI aqueous solution with the concentration ratio of 10% is slowly dripped, and the pH value is adjusted to 7;

in step 2, tris (dibenzylideneacetone) dipalladium pb ₂ dba ₃ As a catalyst and in 5-di-tert-butylphosphine-1 ',3',5' -triphenyl-1 ' H- [1,4 ] ']The reaction is carried out under the action of BippyPhos, and the reaction condition is 110 ℃ for 6 hours without water or oxygen;

in step 3, 10% aqueous hci solution was slowly added dropwise to the methanol mixture, and the mixture was reacted under reflux for 20 minutes.

Further, the compound of claim 2 is prepared from 3-hydroxyacetophenone as a raw material through steps 1 and 2; starting from 3, 5-dihydroxyacetophenone, and preparing the compound of claim 3 through steps 0,1, 2 and 3; starting from 2,3, 4-trihydroxyacetophenone, the compounds according to claim 4 are obtained in steps 0,1, 2, 3.

By adopting the technical scheme, the invention has the following beneficial effects:

1. the invention provides a method for synthesizing, separating and purifying 1-phenyl-3- (4- (pyrimidine-5-ylamino) phenyl) prop-2-en-1-one compounds with extremely high polarity and relatively high synthesis and separation difficulty.

2. Experiments prove that the compound synthesized by the invention has good inhibition effect on the aggregation of the Abeta protein, protects the nerve cell activity by inhibiting the aggregation of the Abeta protein, has the drug activity for inhibiting the aggregation of the Abeta protein, wherein the effect of the compound A-N-5 is obviously better than the effect of positive drugs curcumin and EGCG, and can provide a lead compound for developing Alzheimer's disease drugs.

3. The compounds synthesized according to the present invention have been shown to inhibit cellular iron death by inhibiting cellular lipid peroxidation levels. It is hopeful to further develop the iron death inhibitor for treating the diseases related to cell iron death.

Drawings

FIG. 1 is a schematic diagram of a compound A-C-3 of the present invention ¹ H NMR spectrum;

FIG. 2 is a schematic diagram of the compound A-C-3 of the present invention ¹³ C NMR spectrum;

FIG. 3 is a schematic diagram of the compound A-C-5 of the present invention ¹ H NMR spectrum;

FIG. 4 is a schematic diagram of the compound A-C-5 of the present invention ¹³ C NMR spectrum;

FIG. 5 is a schematic diagram of the compound A-M-1 of the present invention ¹ H NMR spectrum;

FIG. 6 is a diagram of the compound A-M-1 of the present invention ¹³ C NMR spectrum;

FIG. 7 is a schematic diagram of the compound A-M-2 of the present invention ¹ H NMR spectrum;

FIG. 8 is a schematic diagram of the compounds A-M2 according to the invention ¹³ C NMR spectrum;

FIG. 9 is a schematic diagram of the compound A-M-4 of the present invention ¹ H NMR spectrum;

FIG. 10 is a schematic diagram of the compound A-M-4 of the present invention ¹³ C NMR spectrum;

FIG. 11 is a schematic diagram of the compound A-M-5 of the present invention ¹ H NMR spectrum;

FIG. 12 is a diagram of the compound A-M-5 of the present invention ¹³ C NMR spectrum;

FIG. 13 is a schematic diagram of a compound A-N-1 of the present invention ¹ H NMR spectrum;

FIG. 14 is a schematic diagram of the compound A-N-1 of the present invention ¹³ C NMR spectrum;

FIG. 15 is a schematic diagram of the compound A-N-2 of the present invention ¹ H NMR spectrum;

FIG. 16 is a graph of the compound A-N-2 of the present invention ¹³ C NMR spectrum;

FIG. 17 is a graph of the compound A-N-4 of the present invention ¹ H NMR spectrum;

FIG. 18 is a graph of the compound A-N-4 of the present invention ¹³ C NMR spectrum;

FIG. 19 is a diagram of the compound A-N-5 of the present invention ¹ H NMR spectrum;

FIG. 20 is a diagram of the compound A-N-5 of the present invention ¹³ C NMR spectrum;

FIG. 21 is a graph showing the results of an experiment for detecting the activity of 1-phenyl-3- (4- (pyrimidin-5-ylamino) phenyl) prop-2-en-1-one compounds by the ThT method;

FIG. 22 is a graph showing the experimental results of cytotoxicity of 1-phenyl-3- (4- (pyrimidin-5-ylamino) phenyl) prop-2-en-1-one compounds;

FIG. 23 is a graph showing the experimental results of the degree of inhibition of toxicity caused by aggregation of Aβ protein by 1-phenyl-3- (4- (pyrimidin-5-ylamino) phenyl) prop-2-en-1-one compounds at high concentrations;

FIG. 24 is a graph showing the experimental results of the degree of inhibition of toxicity caused by aggregation of Abeta protein by 1-phenyl-3- (4- (pyrimidin-5-ylamino) phenyl) prop-2-en-1-one compounds at low concentrations;

FIG. 25 is a graph showing the experimental results of the extent to which 1-phenyl-3- (4- (pyrimidin-5-ylamino) phenyl) prop-2-en-1-one compounds inhibit the level of lipid peroxidation caused by Aβ protein;

FIG. 26 is a graph of experimental results of the extent to which 1-phenyl-3- (4- (pyrimidin-5-ylamino) phenyl) prop-2-en-1-one compounds inhibit the level of lipid peroxidation induced by RSL-3;

FIG. 27 shows the compounds A-N-5 and Abeta of the present invention _1-42 A 2D model map of protein molecule docking;

FIG. 28 is a 2D model of the molecular docking of compound A-N-5 of the invention with Nrf2 protein.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the drawings and detailed description are only intended to illustrate the invention and are not intended to limit the invention.

Example 1

1-phenyl-3- (4- (pyrimidin-5-ylamino) phenyl) prop-2-en-1-one compounds or pharmaceutically acceptable salts thereof, compounds of formula (I),

wherein R1, R2, R3 and R4 are each hydrogen or hydroxy, R5 and R6 are C-containing _1-16 Alkyl or C _2-16 Alkenyl groups.

The invention provides a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier or excipient. The invention can be singly used or prepared into other clinically available medicines with different dosage forms, wherein the dosage forms comprise powder, injection, capsule, pill, microcapsule, tablet, film, soft capsule, paste, suppository, aerosol, tincture, oral liquid and granule. Pharmaceutically acceptable pharmaceutical excipients including fillers, binders, wetting agents, disintegrants, pH adjusting agents or lubricants and the like may be added according to pharmaceutical pharmacy.

The application of the compound in preparing medicines for preventing and treating Alzheimer's disease and in preparing inhibitors for cell iron death. Compared with the prior known Abeta protein inhibitor with better effect, the 1-phenyl-3- (4- (pyrimidine-5-ylamino) phenyl) prop-2-en-1-one compound provided by the invention has better inhibition effect in vitro and cell models, and can inhibit cell iron death by inhibiting lipid peroxidation. In particular the compound (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (2, 3, 4-trihydroxyphenyl) prop-2-en-1-one, holds promise for further development for the treatment of Alzheimer's disease and diseases associated with cell iron death.

The inhibitor and the Abeta protein are usually beta-folded parts of the protein, the atoms forming hydrogen bond are mainly O, N, F atoms, and simultaneously, the compound with a rigid plane structure and a conjugated structure is easier to combine with the Abeta protein. Both iron death inhibitors ferrostatin-1 and liproxstatin-1, which are screened by high throughput, are aromatic amine structures and diarylamine derivatives are potent inhibitors of iron death. By the aza-s of the diarylaminesThe analogue is modified to achieve good stability and higher reactivity. The lower the dissociation energy of the secondary amine N-H bond in the diarylamine structure, the rate constant k of the reaction of the molecule with lipid peroxy radicals (LOO.) _inh The larger. The inventors have introduced an electron donating group-N (CH) ₃ ) ₂ To reduce dissociation energy of N-H and to make reaction rate constant k _inh The improvement is 1-2 orders of magnitude. Thereby increasing the rate of reaction of the molecule with the lipid peroxy radical.

In addition, the electron donating group removes-N (CH) ₃ ) ₂ May also be N (C _1-16 -alkyl group ₂ Or N (C) _2-16 -alkenyl groups ₂ With the growth of alkyl or alkenyl chain in the electron donating group, the electron donating effect is gradually increased, so that the reaction rate constant is improved, and the preparation method is suitable for changing the side chain of the electron donating group into N (C) _1-16 -alkyl group ₂ Or N (C) _2-16 -alkenyl groups ₂ Is the case in (a).

The invention provides a preparation method of a 1-phenyl-3- (4- (pyrimidine-5-ylamino) phenyl) prop-2-en-1-one compound, which comprises the following steps:

1) Firstly, acetophenone and p-bromobenzaldehyde are catalyzed by 10% KOH aqueous solution in ethanol solution, after the reaction is carried out for 48 to 96 hours at normal temperature, 10% HCI aqueous solution is slowly dripped to adjust the pH to be 7, and the corresponding chalcone compound is obtained after treatment.

2) Then the product of the step 1) is reacted with N2, N2-dimethyl-2, 5-diaminopyrimidine, and palladium catalyst pb is adopted ₂ dba ₃ And is carried out under the action of BippyPhos. The reaction was carried out at 110℃for 6 hours under anhydrous and anaerobic conditions to reduce by-products.

3) If the acetophenone in the step 1) is 3, 5-dihydroxyacetophenone or 2,3, 4-trihydroxyacetophenone, the bromomethyl ether is used for hydroxy protection before the step 1) is carried out.

4) Dissolving 3, 5-dihydroxyacetophenone or 2,3, 4-dihydroxyacetophenone in anhydrous acetone, adding 3 times of potassium carbonate, slowly dropwise adding 3 times of bromomethyl methyl ether under ice bath condition, reflux reacting for 4 hours, cooling to room temperature, and processing to obtain methoxymethyl protected acetophenone.

5) Dissolving the product containing the methoxymethyl protection obtained in the step 2) in methanol, slowly dropwise adding 10% HCI aqueous solution, reacting for 20 minutes under the reflux condition, and processing to obtain a target final product.

Example 2

(E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (3-hydroxyphenyl) prop-2-en-1-one is prepared as follows:

1. preparation of (E) -3- (4-bromophenyl) -1- (3-hydroxyphenyl) prop-2-en-1-one (A-C-3)

Wherein, in the first step of the present invention, 0.27g (2 mmol) of 3-hydroxyacetophenone was added to ethanol (20 mL) and stirred until completely dissolved, followed by sequential addition of KOH aqueous solution (10% w/v,10 mL) and 0.44g (2.4 mmol) of p-bromobenzaldehyde. The mixture was reacted at room temperature for 72h.

After completion of the reaction by TLC, the reaction mixture was pH-adjusted to 7 with aqueous HCl (10% v/v), and extracted 3 times with water and ethyl acetate, respectively. The crude product obtained by distillation under reduced pressure was purified by silica gel column chromatography using ethyl acetate/petroleum ether (gradient elution from 1:10 to 1:3) as an eluent to give 0.25g of (E) -3- (4-bromophenyl) -1- (3-hydroxyphenyl) prop-2-en-1-one (abbreviated as: A-C-3) as a yellow solid in a yield of 42.7%.

A-C-3 is yellow solid powder, HRMS Mass Spectrometry (ESI) m/z: c (C) ₁₅ H ₁₁ BrO ₂ (M ⁺ ) Theoretical calculated value: 302.9976; test value: 303.0014[ M+H ]] ⁺ 。

Analytical data for A-C-3 as shown in FIGS. 1 and 2, compound A-C-3 was determined ¹ H NMR is: (400 MHz, CD) ₃ OD)δ7.68(d,J＝1.0Hz,2H),7.62(d,J＝8.6Hz,2H),7.59-7.52(m,3H),7.45-7.41(m,1H),7.35(t,J＝7.9Hz,1H),7.05(ddd,J＝8.1,2.5,0.9Hz,1H)。

Shown in FIG. 2 ¹³ The C NMR spectrum was taken from the sample, ¹³ c NMR was: (100 MHz, CD) ₃ OD)δ190.65,157.77,143.10,139.20,134.03,131.89,129.90,129.53,124.33,122.56,120.10,119.67,114.46。

2. Preparation of (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (3-hydroxyphenyl) prop-2-en-1-one (A-C-5)

In step two, at N ₂ 18.3mg (0.02 mmol) of Pb2dba3, 40.48mg (0.08 mmol) of Bippyphos, t-amyl alcohol (2 mL), 8.41mg (0.15 mmol) of KOH and 0.02mL of H are added in sequence to a dry flask in an atmosphere ₂ O, after stirring the mixture at room temperature for 20min, 30.3mg of A-C-3 (0.1 mmol) and 11.0mg (0.08 mmol) of N2, N2-dimethyl-2, 5-diaminopyrimidine were added, after which the mixture was refluxed at 102℃for 6h.

After completion of the reaction by TLC, the crude product was obtained by distillation under reduced pressure and purified by silica gel column chromatography using ethyl acetate/petroleum ether (gradient elution 1:5 to 2:1) as eluent to give 13.3mg of a reddish brown viscous liquid, (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (3-hydroxyphenyl) prop-2-en-1-one (abbreviated: a-C-5) in 37.7% yield.

A-C-5 is a reddish brown viscous liquid, HRMS mass spectrometry (ESI) m/z: c (C) ₂₁ H ₂₀ N ₄ O ₂ (M ⁺ ) Theoretical calculated value: 361.1620; test value: 361.1653[ M+H ]] ⁺ The structural formula is as follows:

the analysis data of A-C-5 are shown in FIGS. 3 and 4, and the analysis data of the compound A-C-5 is determined ¹ H NMR is: (700 MHz, DMSO-d 6) δ9.71 (s, 1H), 8.23 (m, 3H), 7.57 (m, 5H), 7.35 (m, 2H), 6.99 (s, 1H), 6.73 (s, 2H), 3.10 (s, 6H).

¹³ C NMR was: (175 MHz, DMSO-d 6) delta 188.80,159.37,157.73,153.82,149.30,144.70,139.78,131.07,129.79,124.97,124.21,119.81,119.31,117.10,114.53,113.13,37.05。

Example 3

(E) The preparation route of 1- (3, 5-dihydroxyphenyl) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) prop-2-en-1-one (a-M-5) is:

1. preparation of 1- (3, 5-bis (methoxymethoxy) phenyl) ethan-1-one (A-M-1)

In step one of the preparation process of the present invention, 0.76g (5 mmol) of 3, 5-dihydroxyacetophenone and 1.86g (15 mmol) of K2CO3 were stirred in anhydrous acetone (50 mL) until completely dissolved, 2.48g (20 mmol) of bromomethyl ether was slowly added dropwise under ice bath conditions, the reaction mixture was cooled on ice bath for 30min, and then refluxed at 60℃for 4h. After completion of the reaction by TLC, the reaction mixture was cooled to room temperature, and extracted 3 times with water and ethyl acetate, respectively. The crude product obtained by distillation under reduced pressure was purified by silica gel column chromatography using ethyl acetate/petroleum ether (1:5) as an eluent to give 0.42g of 1- (3, 5-bis (methoxymethoxy) phenyl) ethan-1-one (abbreviated as: A-M-1) as a colorless liquid.

A-M-1 is colorless liquid HRMS mass spectrometry (ESI) M/z: c (C) ₁₂ H ₁₆ O ₅ (M ⁺ ) Theoretical calculated value: 241.1031; test value: 241.1075[ M+H ]] ⁺ 。

FIGS. 5 and 6 show the results of analysis of the compound A-M-1, A-M-1 ¹ H NMR is:

(400MHz,CDCl ₃ )δ7.27(d,J＝2.3Hz,2H),6.94(t,J＝2.3Hz,1H),5.20(s,4H),3.49(s,6H),2.57(s,3H)。

¹³ c NMR was: (100 MHz, CDCl) ₃ )δ197.44,158.38,139.24,109.55,109.52,94.53,56.19,26.76。

2. Preparation of (E) -1- (3, 5-bis (methoxymethoxy) phenyl) -3- (4-bromophenyl) prop-2-en-1-one (A-M-2)

In step one of the preparation process of the present invention, 0.48g (2 mmol) of A-M-1 was added to ethanol (20 mL) and stirred until completely dissolved, followed by the sequential addition of aqueous KOH (10% w/v,10 mL) and 0.44g (2.4 mmol) of p-bromobenzaldehyde. The mixture was reacted at room temperature for 72h. After completion of the reaction by TLC, the pH of the reaction mixture was adjusted to 7 with aqueous HCl (10% v/v), and the extraction was performed 3 times with water and ethyl acetate, respectively. The crude product obtained by distillation under reduced pressure was purified by silica gel column chromatography using ethyl acetate/petroleum ether (gradient elution from 1:10 to 1:3) as an eluent to give 0.70g of (E) -1- (3, 5-bis (methoxymethoxy) phenyl) -3- (4-bromophenyl) prop-2-en-1-one (abbreviation: A-M-2) as a pale yellow solid in 86.6% yield.

A-M-2 is a pale yellow solid, HRMS Mass Spectrometry (ESI) M/z: c (C) ₁₉ H ₁₉ BrO ₅ (M ⁺ ) Theoretical calculated value: 407.0449; test value: 407.0482[ M+H ]] ⁺ 。

FIGS. 7 and 8 are the results of analysis of the compound A-M-2, A-M-2 ¹ H NMR is: (400 MHz, CDCl) ₃ )δ7.81(d,J＝15.5Hz,2H),7.66-7.40(m,5H),6.65(s,1H),6.58(s,1H),5.23(s,4H),3.49(s,6H)；

¹³ C NMR was: (100 MHz, CDCl) ₃ )δ191.74,166.32,163.84,143.16,133.69,132.28,131.32,129.89,125.02,120.86,114.87,108.36,104.01,94.06,56.45。

3. Preparation of (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (3, 5-di (methoxymethoxy) phenyl) prop-2-en-1-one (A-M-4)

In step two of the production method of the present invention, 18.3mg (0.02 mmol) of Pb was successively added to a dry flask in an N2 atmosphere ₂ dba ₃ 40.48mg (0.08 mmol) Bippyphos, t-amyl alcohol (2 mL), 8.41mg (0.15 mmol) KOH and 0.02mL H ₂ O, after stirring the mixture at room temperature for 20min, 40.7mg of A-M-2 (0.1 mmol) and 11.0mg (0.08 mmol) of N2, N2-dimethyl-2, 5-diaminopyrimidine were added, after which the mixture was refluxed at 102℃for 6h. After completion of the reaction by TLC, the crude product was distilled under reduced pressure and purified by silica gel column chromatography using ethyl acetate/petroleum ether (gradient elution 1:5 to 2:1) as eluent to give 6.00mg of reddish brown viscous liquid E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (3, 5-dihydroxyphenyl) prop-2-en-1-one (abbreviation: A-M-4), yield 12.9%.

FIGS. 9 and 10 show the results of analysis of Compound A-M-4, A-M-4 ¹ H NMR is: (400 mhz, dmso-d 6) δ8.30 (s, 2H), 8.26 (s, 1H), 7.78-7.47 (m, 4H), 7.37 (d, j=2.2 hz, 2H), 6.93 (s, 1H), 6.77 (d, j=8.6 hz, 2H), 5.28 (s, 4H), 3.41 (s, 6H), 3.14 (s, 6H).

¹³ C NMR was: (100 MHz, DMSO-d 6) delta 188.53,159.77,158.42,154.19,149.81,145.67,140.87,131.68,125.33,124.57,117.22,113.51,109.62,109.14,94.52,56.26,37.44.

4. Preparation of (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (3, 5-dihydroxyphenyl) prop-2-en-1-one (A-M-5)

In step three of the preparation process of the present invention, methoxymethyl protected 23.2mg (0.05 mmol) of A-M-4 was added to methanol and 2mL of HCl (10% v/v in water) was slowly added dropwise. At N ₂ The reaction mixture was refluxed for 30min in the atmosphere. The reaction was completed by TLC and cooled to room temperature. After concentrating the crude product under reduced pressure, the crude product was washed with 3 x 50ml ethyl acetate, filtered and dried to give 7.50mg of (E) -1- (3, 5-dihydroxyphenyl) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) prop-2-en-1-one (abbreviated: a-M-5) as a red powdery solid in 39.8% yield.

A-M-5 is a red powdered solid, HRMS Mass Spectrometry (ESI) M/z: c (C) ₂₁ H ₂₀ N ₄ O ₃ (M ⁺ ) Theoretical calculated value: 377.1569; test value: 377.1609[ M+H ]] ⁺ The structural formula is as follows:

FIGS. 11 and 12 show the results of A-M-5 analysis, compound A-M-5 ¹ H NMR is: (700 mhz, dmso-d 6) δ8.36 (s, 2H), 7.64 (d, j=8.3 hz, 2H), 7.58 (d, j=15.4 hz, 1H), 7.43 (d, j=15.4 hz, 1H), 6.90 (s, 2H), 6.80 (d, j=8.2 hz, 2H), 6.49 (s, 1H), 3.16 (s, 6H).

¹³ C NMR was: (175 MHz, DMSO-d 6) delta 189.36,159.10,157.74,153.20,149.04,144.78,140.74,131.32,125.63,125.02,117.94,113.87,107.20,106.75,37.72.

Example 4

(E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (2, 3, 4-trihydroxyphenyl) prop-2-en-1-one was prepared as follows:

1. preparation of 1- (2-hydroxy-3, 4-bis (methoxymethoxy) phenyl) ethan-1-one (A-N-1)

In step one of the preparation process according to the invention, 0.84g (5 mmol) of 2,3, 4-trihydroxyacetophenone and 1.86g (15 mmol) of K are reacted ₂ CO ₃ 2.48g (20 mmol) of bromomethyl ether was slowly added dropwise under ice-bath conditions with stirring to complete dissolution in anhydrous acetone (50 mL), and the reaction mixture was cooled on an ice bath for 30min and then refluxed at 60℃for 4h. After completion of the reaction by TLC, the reaction mixture was cooled to room temperature, and extracted 3 times with water and ethyl acetate, respectively. The crude product obtained by distillation under reduced pressure was purified by silica gel column chromatography using ethyl acetate/petroleum ether (1:5) as an eluent to give 0.41g of 1- (2-hydroxy-3, 4-di (methoxymethoxy) phenyl) ethan-1-one (abbreviated as: A-N-1) as a white solid in a yield of 32.5%.

A-N-1 is a white solid, HRMS Mass Spectrometry (ESI) m/z: c (C) ₁₂ H ₁₆ O ₆ (M ⁺ ) Theoretical calculated value: 257.0980; test value: 257.1011[ M+H ]] ⁺ 。

FIGS. 13 and 14 are analysis results of A-N-1, compound A-N-1 ¹ H NMR is: (400 MHz, CDCl) ₃ )δ7.48(d,J＝9.1Hz,1H),6.72(d,J＝9.1Hz,1H),5.28(s,2H),5.19(s,2H),3.64(s,3H),3.51(s,3H),2.57(s,3H).

¹³ C NMR was: (100 MHz, CDCl) ₃ )δ203.17,157.12,156.04,133.54,126.78,115.56,105.86,97.74,94.33,56.99,56.21,37.25,26.20.

2. Preparation of (E) -3- (4-bromophenyl) -1- (2-hydroxy-3, 4-di (methoxymethoxy) phenyl) prop-2-en-1-one (A-N-2)

In step one of the present invention, 0.51g (2 mmol) of A-N-1 was added to ethanol (20 mL) and stirred until completely dissolved, followed by the sequential addition of aqueous KOH (10% w/v,10 mL) and 0.44g (2.4 mmol) of p-bromobenzaldehyde. The mixture was reacted at room temperature for 72h. After completion of the reaction by TLC, the pH of the reaction mixture was adjusted to 7 with aqueous HCl (10% v/v), and the extraction was performed 3 times with water and ethyl acetate, respectively. The crude product obtained by distillation under reduced pressure was purified by silica gel column chromatography using ethyl acetate/petroleum ether (gradient elution from 1:10 to 1:3) as an eluent to give 0.55g of (E) -3- (4-bromophenyl) -1- (2-hydroxy-3, 4-di (methoxymethoxy) phenyl) prop-2-en-1-one (abbreviated as: A-N-2) as a yellow solid in 65.1% yield.

A-N-2 is a yellow solid, HRMS Mass Spectrometry (ESI) m/z: c (C) ₁₉ H ₁₉ BrO ₆ (M ⁺ ) Theoretical calculated value: 423.0399; test value: 423.0435[ M+H ]] ⁺ 。

FIGS. 15 and 16 show the analysis results of A-N-2, ¹ h NMR is: (400 MHz, CDCl) ₃ )δ13.20(s,1H),7.81(d,J＝15.5Hz,1H),7.64(d,J＝9.2Hz,1H),7.60-7.43(m,5H),6.76(d,J＝9.1Hz,1H),5.26(d,J＝31.5Hz,4H),3.59(d,J＝55.2Hz,6H).

¹³ C NMR was: (100 MHz, CDCl) ₃ )δ192.37,158.69,156.57,143.48,134.08,133.61,132.31,129.96,126.09,125.15,120.80,116.04,106.17,98.05,94.64,57.32,56.55.

3. Preparation of (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (2-hydroxy-3, 4-di (methoxymethoxy) phenyl) prop-2-en-1-one (A-N-4)

In step two of the present invention, in N ₂ 18.3mg (0.02 mmol) of Pb was successively added to the dry flask in the atmosphere ₂ dba ₃ 40.48mg (0.08 mmol) of Bippyphos, t-amyl alcohol (2 mL), 8.41mg (0.15 mmol) of KOH and 0.02mL of H2O, after stirring the mixture at room temperature for 20min, 42.3mg of A-N-2 (0.1 mmol) and 11.0mg (0.08 mmol) of N2, N2-dimethyl-2, 5-diaminopyrimidine are added, after which the mixture is refluxed for 6h at 102 ℃. After completion of the reaction by TLC, the crude product was distilled under reduced pressure and purified by silica gel column chromatography using ethyl acetate/petroleum ether (gradient elution 1:5 to 2:1) as eluent to give 19.6mg of (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (2-hydroxy-3, 4-bis (methyl) as a reddish brown viscous liquidOxymethoxy) phenyl) prop-2-en-1-one (abbreviation: A-N-4), yield was 41.7%.

A-N-4 is reddish brown viscous liquid, HRMS mass spectrum (ESI) m/z: c (C) ₂₅ H ₂₈ N ₄ O ₆ (M ⁺ ) Theoretical calculated value: 481.2081; test value: 481.2042[ M+H ]] ⁺ The molecular formula is shown in the specification.

FIGS. 17 and 18 show the results of analysis of A-N-4, compound A-N-4 ¹ H NMR is: (700 MHz, CD) ₃ OD)δ8.23(s,1H),8.21(s,1H),7.83(d,J＝9.2Hz,1H),7.78(d,J＝15.2Hz,1H),7.62-7.55(m),7.53(d,J＝15.2Hz,1H),7.29(d,J＝8.5Hz,1H),6.90-6.72(m,3H),5.41-5.20(m,2H),5.18-4.99(m,2H),3.62-3.44(m,6H),3.17(s,3H),3.15(s,3H).

¹³ C NMR was: (175 MHz, CD) ₃ OD)δ194.15,155.21,154.61,150.99,148.42,146.87,134.90,132.15,129.04,127.41,126.12,123.79,117.26,116.38,114.78,114.52,99.31,99.02,95.85,95.71,94.12,57.49,56.73,37.71,37.68.

4. Preparation of (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (2, 3, 4-trihydroxyphenyl) prop-2-en-1-one (A-N-5)

In step three of the preparation process of the present invention, methoxymethyl protected 24mg (0.05 mmol) of A-N-4 was added to methanol and 2mL of HCl (10% v/v in water) was slowly added dropwise. The reaction mixture was refluxed for 30min in an N2 atmosphere. The reaction was completed by TLC and cooled to room temperature. After concentrating the crude product under reduced pressure, the crude product was washed with 3 x 50ml ethyl acetate, filtered and dried to give 10.7mg of (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (2, 3, 4-trihydroxyphenyl) prop-2-en-1-one (abbreviated as a-N-5) as a red powder in 55.2% yield.

A-N-5 is a red solid powder, HRMS Mass Spectrometry (ESI) m/z: c (C) ₂₁ H ₂₀ N ₄ O ₄ (M ⁺ ) Theoretical calculated value: 393.1518; test value: 393.1559[ M+H ]] ⁺ The structural formula is as follows:

FIGS. 19 and 20 show the results of analysis of A-N-5, compound A-N-5 ¹ H NMR is: (700 mhz, dmso-d 6) delta 13.70 (s, 1H), 8.35 (s, 1H), 8.34 (s, 1H), 7.69 (m, j=8.1 hz, 3H), 7.40 (d, j=8.1 hz, 1H), 7.17-7.07 (m, 2H), 6.90-6.81 (m, 2H), 6.56 (d, j=8.8 hz, 1H), 6.45 (d, j=8.9 hz, 1H), 3.16 (s, 6H).

¹³ C NMR was: (175 MHz, DMSO-d 6) delta 192.39,154.09,152.85,149.33,144.79,132.92,131.72,128.73,128.41,125.51,124.92,123.74,122.62,117.84,116.47,114.79,114.13,113.79,108.10,37.68.

Example 5

The effect of 1-phenyl-3- (4- (pyrimidin-5-ylamino) phenyl) prop-2-en-1-one compounds prepared in examples 2,3,4 of the present invention on the aggregation level of Abeta protein was examined by the ThT method.

The ThT detection method comprises the following steps: 1) Will Aβ _1-42 The protein was dissolved in 1mL of 1, 3-hexafluoro-2-propanol (HFIP) and N was used ₂ Blow-dried and then prepared as a 1mM DMSO solution.

2) Will Aβ _1-42 Protein (25. Mu.M) was placed in 10. Mu.M phosphate buffer (pH 7.4), inhibitor (0, 10. Mu.M, 25. Mu.M) was added at different concentrations, each sample was diluted to 100. Mu.L with phosphate buffer, incubated at 37℃for 7 days, and corresponding concentrations of compounds A-N-5, A-M-5, A-C-5 and the positive drugs curcumin and EGCG were added to the corresponding wells, respectively, and three groups were set in parallel.

3) Samples were placed in 96-well plates and thioflavin-T (0.5 mM) was added using a fluorolabeller (λex=440 nm; λem=485 nm) was used for measurement of fluorescence intensity. Percent inhibition of aggregation was calculated using the formula (1-Fi/Fc) ×100%, where Fi and Fc are the fluorescence intensities obtained after subtracting the background with and without inhibitor, respectively. Each experiment was independently repeated at least 3 times.

The results of the assay are shown in FIG. 21, where the compounds tested have inhibitory activity against Abeta protein aggregation: compound a-N-5>A-M-5>A-C-5, wherein the inhibition of compound a-N-5 is 99.1% to 95.8% higher than positive control EGCG (75.9%) and curcumin (59.4%). The results show that the three compounds can well inhibit the aggregation of Abeta protein, and the target direction is expected to improve the disease state and even prevent the disease state from developing while delaying the disease state of senile dementia. The compound A-N-5 has extremely high inhibition effect on Abeta protein aggregation, and is hopeful to be further developed for treating Alzheimer's disease.

Example 6

The effects of cytotoxicity of the 1-phenyl-3- (4- (pyrimidin-5-ylamino) phenyl) prop-2-en-1-one compounds prepared in examples 2,3,4 and the degree of inhibition of toxicity caused by the aggregation of aβ protein by the compounds were examined by cell experiments.

1. Cell assay method to detect cytotoxicity: 1) HT22 cells were grown to log phase at 5X 10 ³ Cells/100 μl/well were plated into 96-well plates, and after 24 hours of cell adhesion, cells were treated with different concentrations of compound for 24 hours, with 6 duplicate wells set for each concentration. 2) The MTS method (MTS assay; promega) was used to determine cell viability, 20. Mu.L MTS was added to each well, incubated for 4h in the dark and absorbance at 490nm and 560nm was measured on a fluorescent microplate reader. 3) Cell viability was proportional to the absorbance of each well and a negative control group was set for normalization. Mean ± standard deviation (mean ± standard) of cell viability was calculated for each group of at least three wells.

As shown in FIG. 22, the toxicity results of the compounds are shown in FIG. 22, and the test compounds have no cytotoxicity at a concentration of 40. Mu.M, and the activity test concentration of the compounds is lower than 20. Mu.M, so that the compounds can be further subjected to the activity test.

2. The experimental method for detecting the Abeta protein aggregation cells comprises the following steps:

1) SH-SY5Y cells were grown to log phase at 2X 10 ⁴ Cells/100. Mu.L/well were plated in 96-well plates, after 24 hours of cell adhesion, the cells were treated with different concentrations of compounds, 6 multiplex wells were placed at each concentration, 2) 4 hours later, and 15. Mu. M A. Beta. Was added _1-42 The protein was then placed in an incubator for 48h. Cell viability was determined using the MTS assay (MTS assay; promega) according to the manufacturer's instructions, 20. Mu.L MTS was added to each well and absorbance at 490nm and 560nm was measured on a fluorescence microplate reader after incubation for 4 hours in the absence of light. 3) Cell viability was proportional to absorbance per well and 6 no-compound addition with only aβ _1-42 Protein multiplex pore is negative pairIn the group, compound EGCG and curcumin were set as positive controls. Mean ± standard deviation (mean ± standard) of cell viability was calculated for each group of at least three wells.

The results of the activity of the compounds are shown in FIGS. 23 and 24, and it is understood from the figures that the compound A-N-5 can help cells almost completely inhibit toxicity caused by aggregation of Abeta proteins at concentrations of 20. Mu.M and 10. Mu.M. Under the action of all tested concentration ranges, the activity of the composition is superior to that of positive medicines curcumin and EGCG. The concentration was reduced to 1. Mu.M and 0.5. Mu.M, and the effect of activity was still shown to be superior to that of the positive control EGCG. Compared with the trihydroxy chalcone compound (E) -3- (4-bromophenyl) -1- (2, 3, 4-trihydroxy phenyl) prop-2-en-1-one (A-N-3), the A-N-5 has better activity effect under the same concentration. Therefore, the compound A-N-5 reduces nerve cell death caused by Abeta protein aggregation by inhibiting Abeta protein aggregation, has obviously improved activity compared with chalcone compounds, and is hopeful to be further developed for treating Alzheimer's disease.

Example 7

The effect of the compounds of examples 2,3,4 of the present invention on the extent of inhibition of cellular iron death was examined by cell experiments.

1. Method for detecting influence on inhibition degree of iron death caused by erastin by cell experiment:

1) HT22 cells were grown to log phase at 5X 10 ³ Cells/100. Mu.L/well were plated in 96-well plates, and after 24 hours of cell adhesion, cells were treated with the compounds prepared in examples 2,3, and 4 at different concentrations, 6 duplicate wells were set for each concentration, and after 4 hours of incubation, iron death activator erastin at a concentration of 10. Mu.M was added.

2) The MTS method (MTS assay; promega) was used to determine cell viability, 20. Mu.L MTS was added to each well, incubated for 4h in the dark and absorbance at 490nm and 560nm was measured on a fluorescent microplate reader.

3) Cell viability was proportional to the absorbance of each well, and 6 compound-free multiplexed wells with erastin protein alone were set as negative controls. Mean ± standard deviation (mean ± standard) of cell viability was calculated for each group of at least three wells.

2. Method for cell experiments to examine the effect on the extent of inhibition of iron death by RSL-3:

the procedure was followed as described above, except that 500nM RSL-3 was used instead of erastin.

The activity results of the compounds are shown in table 1 below:

TABLE 1

Remarks: 1) The values in the table are expressed as mean ± standard deviation of three experiments;

2)IC ₅₀ and for half inhibition concentration, N represents that the inhibition rate of the compound is less than 10% at the concentration of 10 mu M, namely the compound is inactive.

The three compounds of the invention have better effect of inhibiting iron death in cell models of two different iron death initiators, and half inhibition rate is between 2.65 and 0.39 mu m. The control drug EGCG did not have the effect of inhibiting iron death.

Example 8

The effect of compounds on the extent of inhibition of lipid peroxidation levels was examined by cell experiments.

1. A method for detecting the effect of a compound on the extent of inhibition of aβ protein-induced lipid peroxidation by a cell assay: 1) SH-SY5Y cells were grown to log phase at 2X 10 ⁴ Cells/100 μl/well were plated into 96-well plates, and after 24 hours of cell adhesion, cells were treated with different concentrations of the compounds of examples 2,3,4, each concentration being set with 6 duplicate wells. 2) After 4h 20 mu M A beta is added _1-42 The protein was then placed in an incubator for 48h. Setting 6 compounds without adding Abeta _1-42 Protein multiplex wells, 61 mM diethyl maleate (DEM) multiplex wells, 6 multiplex wells without any treatment served as control. 3) Then discarding the culture medium in the holes, adding 100 mu LPBS for cleaning, discarding, adding 20 mu L of trypsin into each hole to suspend the cells for 2min, adding 80 mu L of culture medium to stop the action of trypsin, transferring six compound holes of the same group into a 15ml centrifuge tube for centrifugation, discarding the supernatant, adding 500 mu LPBS for resuspension, transferring into a special centrifuge tube of a flow cytometer,adding 1 μm fluorescence indicator BODIPY-C11 ^581/591 Incubate for 30min in the dark. 4) Analysis was performed using a flow cytometer (λex=488 nm; λem=525±25 nm), followed by data analysis and processing using Flowjo (V10) software. The parallel test was performed three times and the results are shown in fig. 25.

2. A method for testing the effect of a compound on the extent of inhibition of lipid peroxidation caused by cellular iron death by a cell assay:

1) HT22 cells were grown to log phase at 5X 10 ³ Cells/100. Mu.L/well were plated into 96-well plates, and after 24 hours of cell adhesion, cells were treated with different concentrations of the compounds prepared in examples 2,3, and 4, each concentration being set with 6 duplicate wells.

2) After 4h 10 μm RSL3 protein was added, followed by incubation in an incubator for 48h. 6 compound-free compound-only RSL3 protein-coated wells, 61 mM diethyl maleate (DEM) coated wells, and 6 untreated coated wells were used as control groups.

3) Removing culture medium in the wells, adding 100 μLPBS, cleaning, removing, adding 20 μL trypsin into each well to suspend cells for 2min, adding 80 μL culture medium to stop trypsin action, transferring six multiple wells of the same group into 15ml centrifuge tube, centrifuging, removing supernatant, adding 500 μLPBS, re-suspending, transferring into special centrifuge tube of flow cytometer, adding fluorescent indicator BODIPY-C11 with concentration of 1 μM ^581/591 Incubate for 30min in the dark.

4) Analysis was performed using a flow cytometer (λex=488 nm; λem=525±25 nm), followed by data analysis and processing using Flowjo (V10) software. The parallel test was performed three times and the results are shown in fig. 26.

The Abeta protein and the RSL-3 can obviously improve the lipid peroxidation degree of cells, and the compound A-N-5 can reduce the lipid peroxidation level to the level of normal cells, so that the series of compounds have the function of reducing the lipid peroxidation level of cells.

Experimental example 9

Compound and protein molecular butt joint to explore molecular action mechanism

1) Compound structure and protein structure introduction and pretreatment

All Molecular structures are drawn by using ChemDraw Professional 15.0.0 software, converted into 3D structures by using Chem3D 15.0 software and saved as a. Mol2 format, a File-New-Molecular Window is clicked in Discovery Studio 3.1 (DS) software, small Molecules-Prepare or Filter Ligands-preparation interfaces are clicked in a toolbar, and a flow parameter setting interface is opened; setting Input links parameters as structures, performing processing on All small molecules, and setting other parameters to default values; clicking Run task.

Aβ download from protein Crystal Structure database (PDB) (http:// www.rcsb.org /) _1-42 Protein crystal structure PDB format file (code: 1 IYT). Opening downloaded Aβ from File in Discovery Studio 3.1 _1-42 And selecting and deleting water molecules in the system view, and sequentially clicking Macromolecules-preparation Protein-Clean Protein in the toolbar to perform Protein optimization treatment. Then, setting the flexibility of amino acid, clicking Recrptor Ligand Interactions-Ligand Interactions in sequence, displaying the amino acid with interaction with the ligand, selecting Define and Edit Binding Site and From Current Selection in a display interface, obtaining a docking Site, and adjusting the coordinate and the size through Attributes of SBD-Site-Sphere.

2) Flexible docking of proteins

And clicking View-Explorers-Protocols in the menu bar in sequence, clicking Receptor Ligand Interactions-Dock liquids in the popped Protocols in sequence, and entering flexible docking. In the parameter setting column, click Input Typed Protein Molecule, select aβ _1-42 Is a receptor protein. Clicking on Input Ligands selects all ligand molecules that were introduced in advance. Click Input Site Sphere, select binding-Sphere, specify active site. Click Generate Ligand Conformations sets the docking parameter, conformation Method selects BEST. Max bits to Save set 3,Parallel Processing to True, TOP bits to 10,Random conformations to 10,Orientations to Refine to 10. And clicking Run to obtain a butt joint result.

3) Analysis and processing of docking results

In the taskbar, clicking View Results to display Receptor ligand docking conformation, clicking Receptor-Ligand Interactions-View interfaces-Analyze Ligand Poses in the taskbar in turn, clicking Input Receptor and Input interfaces to select corresponding docking proteins and ligand conformations respectively, and clicking Run running tasks with the rest parameters as default values. Click View Statistical Information looks at the amino acid residues that interact with the ligand molecule. In the table window of the docking result, set-CDOCKER INTERACTION ENERGY as Top, set Value as 1, select Apply filter to each group of identical values, and screen the docking conformation with highest score of-CDOCKER INTERACTION ENERGY in the docking result.

The results of a series of activity experiments show that the synthesized target compounds have better activities of inhibiting Abeta protein aggregation and cell iron death, and in order to explore the action mechanism of the compounds, abeta protein and iron death, a design thought is provided for the structural optimization of the next step, and the interaction of the molecules and the proteins is explored by selecting a trihydroxy compound A-N-5.

The results of molecular docking of A-N-5 with Abeta protein (PDB: 1 IYT) are shown in FIG. 27, and the interaction energy of A-N-5 with protein is-38.0395 kcal/mol, and the results of analysis of docking are that the interaction of A-N-5 with protein is mainly the hydrogen bonding effect of amino acid Lys16 with hydroxyl group and tertiary amine on benzene ring, pi-alkyl and pi-ion effect of two benzene rings connected with diarylamine, the hydrogen bonding effect of Gln15 with carbonyl group, pi-pi T type effect of Phe19 with left benzene ring and the hydrogen bonding effect of Phe20 with hydroxyl group. The interaction of the analytical compound with the protein is readily found in a variety of ways and more hydrogen bonding is formed by the interaction of A-N-5 with the protein. In addition, the action binding position of A-N-5 and Abeta protein is positioned in the core zipper area of Abeta protein, and according to the literature, the core zipper area of Abeta protein is positioned in amino acids 16-21 of the protein, which is a key action site for inhibiting Abeta protein aggregation, and the reason that the synthetic compound can well inhibit Abeta protein aggregation is also explained.

According to the design thought of the target point of the compound, the activity of inhibiting cell iron death is achieved by inhibiting lipid peroxidation, and nuclear factor erythroid 2 related factor 2 (Nrf 2) is a key regulator of antioxidant reaction. In normal cells, nrf2 is released from Keap1 protein binding and transferred to the nucleus under oxidative or electrophilic effects. In the nucleus, nrf2 transcribes Antioxidant Response Element (ARE) -dependent genes to balance oxidative mediators and maintain cellular redox homeostasis. Thus, nrf2 (3 WN 7) protein was selected for molecular docking with compound A-N-5, and the mechanism of action of its interaction on inhibiting cell iron death was explored.

As a result, as shown in FIG. 28, the interaction energies of the compounds A-N-5 and the Nrf2 protein were-57.8726 kcal/mol, respectively. As a result of analysis, the interaction between A-N-5 and the protein is mainly pi-ion interaction between amino acid Arg and a left benzene ring, and hydrogen bonding interaction between Asn and Asp and a secondary amine and a right benzene ring. The compound A-N-5 has more hydrogen bonding effect with protein, so that the compound A-N-5 has stronger interaction force, and the reason that the compound A-N-5 has better activity of inhibiting lipid peroxidation and cell iron death is explained.

The compound has good inhibition effect on the aggregation of Abeta protein and can inhibit cell iron death, wherein the effect of the compound A-N-5 is obviously better than that of positive medicines curcumin and EGCG, and the compound is hopefully further developed to treat Alzheimer disease and the like or be used as an iron death inhibitor to treat diseases related to cell iron death. The research of molecular docking shows that the compound A-N-5, the Abeta protein and the Nrf2 protein have more hydrogen bonding effect and stronger interaction force, and the reason that the compound activity is better is explained.

The foregoing examples merely illustrate embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. 1-phenyl-3- (4- (pyrimidin-5-ylamino) phenyl) prop-2-en-1-one compounds or pharmaceutically acceptable salts thereof, characterized in that the compounds of formula (I),

wherein R1, R2, R3 and R4 are each hydrogen or hydroxy, R5 and R6 are each C _1-16 Alkyl or C _2-16 Alkenyl groups.

2. A compound according to claim 1, wherein R1 is hydrogen, R2 is hydroxy, R3 is hydrogen, R4 is hydrogen, R5 is methyl, R6 is methyl, and the compound is (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (3-hydroxyphenyl) prop-2-en-1-one.

3. A compound according to claim 1, wherein R1 is hydrogen, R2 is hydroxy, R3 is hydrogen, R4 is hydroxy, R5 is methyl, R6 is methyl, and the compound is (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (3, 5-dihydroxyphenyl) prop-2-en-1-one.

4. A compound according to claim 1, wherein R1 is hydroxy, R2 is hydroxy, R3 is hydroxy, R4 is hydrogen, R5 is methyl, R6 is methyl, and the compound is (E) -3- (4- ((2- (dimethylamino) pyrimidin-5-yl) amino) phenyl) -1- (2, 3, 4-trihydroxyphenyl) prop-2-en-1-one.

5. Use of a compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention and treatment of alzheimer's disease.

6. Use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, for the preparation of an inhibitor of cellular iron death.

7. A pharmaceutical composition comprising a compound according to any one of claims 1-4 and a pharmaceutically acceptable carrier or excipient.

8. A process for the preparation of a compound according to claims 1-4, comprising the steps of:

9. The method for preparing a compound according to claim 8, wherein if acetophenone in the second step is 3, 5-dihydroxyacetophenone or 2,3, 4-trihydroxyacetophenone, step 0 is further included before step 1: reacting polyhydroxy acetophenone with bromomethyl methyl ether, and treating to obtain methoxymethyl-protected acetophenone;

10. The method for preparing a compound according to claim 9, wherein in the step 0,3, 5-dihydroxyacetophenone or 2,3, 4-trihydroxyacetophenone is dissolved in anhydrous acetone, 3 times equivalent potassium carbonate is added, 3 times equivalent bromomethyl ether is slowly added dropwise under the ice bath condition, then reflux reaction is carried out for 4 hours, and after cooling to room temperature, methoxymethyl-protected acetophenone is obtained by treatment;

in the step 2, tris (dibenzylideneandene acetone) dipalladium pb2dba3 is used as a catalyst, and is reacted under the action of 5-di-tert-butylphosphine-1 ',3',5' -triphenyl-1 ' H- [1,4' ] bipyrazole BippyPhos under the reaction conditions of 110 ℃ and 6h without water and oxygen;

11. The method for preparing the compound according to claim 9, wherein the compound according to claim 2 is prepared by using 3-hydroxyacetophenone as a raw material through steps 1 and 2; starting from 3, 5-dihydroxyacetophenone, and preparing the compound of claim 3 through steps 0,1, 2 and 3; starting from 2,3, 4-trihydroxyacetophenone, the compounds according to claim 4 are obtained in steps 0,1, 2, 3.